There are several mechanisms by which virus can cause persistent infection. These include the presence of endogenous interferon, defective interfering (DI) particles, integrated genomes, and-temperature sensitive mutants. To understand the mechanism of persistent infection in congenital rubella we approached the problem in the following manner. We infected Vero 76 cells with rubella virus at a lower multiplicity of infection and isolated cell clones which support restricted replication of virus without a cytopathic effect. A cell line was derived from Vero 76 cells which is persistently infected after a year of infection. The cells shed low levels of virus and are morphologically different from the parent cell. Northern blot analysis of the total RNA isolated from the persistently infected cells showed that there is marked decrease in the level of 40S genomic and 24S subgenomic viral RNA. In addition, a new size class of RNAs ranging from 1.2 to 1.8 kb was present in these cells. A cDNA library of total poly A-positive RNA from the persistently-infected cells was constructed. Several cDNA clones were isolated from this library using rubella cDNA as a probe. Nucleotide sequence of several cDNA clones is being determined. Analysis of nucleotide sequence will give an insight as to how DI RNAs are generated.